Optical techniques that identify and measure biological cells and macromolecules frequently require fluorescent or enzymatic labels as well as a means of isolating or separating analytes. Surface plasmon resonance (“SPR”) techniques separate analytes contained in complex mixtures through the use of specific capture ligands, usually antibodies, bonded to a metallic surface in contact with a dielectric. Light of a specific wavelength striking the metal/dielectric interface at a specific angle can support a rapidly decaying wave phenomenon (surface plasmon) if there is a means of matching the momentum (K-vector) of the light with that of the loosely bound electrons at the metal/dielectric interface. When this resonance energy transfer occurs, the intensity of the light reflected from the metal surface decreases markedly. This resonance phenomenon is quite sharp—on the order of a few millidegrees—and the incident angle is extremely sensitive to the refractive index at the surface of the metal substrate. In the present case, a diffraction grating immediately below a thin layer of metal provides the momentum matching mechanism.
Typically, antibodies bound to the metal surface are used to capture specific analyte molecules present in a complex sample mixture which flows over the metal surface. This highly specific immunochemical process results in specific analyte molecules being bound to well-identified regions of the metal substrate without the necessity of physical compartmentalization of the fluid. For each captured analyte, the magnitude of the change in the resonant angle is proportional to the mass of analyte captured in each region.
With appropriately designed accommodations, an SPR analyzer can be made to capture living cells by surface antigens normally expressed on the surface of the cells. In this manner, specific cell types, distinguished by their surface antigens can be isolated and captured on a metal surface. Cells captured in an SPR analyzer in this manner can be activated by contact with suitable mitogens.
Capture antibodies for various cell secretions (cytokines) can be spotted on the surface in order to immobilize the secreted cytokines. Cytokines are relatively small molecules and the amount of a particular cytokine secreted by a single cell is typically too small to be detected by SPR resonance angle shifts.
It has been observed that energy from surface plasmons can be out-coupled and absorbed by fluorophore molecules in close proximity to the metal surface (see Lakowicz, J. R., 2006, “Plasmonics in Biology and Plasmon-Controlled Fluorescence”, DOI 10.1007/s 11468-005-9002-3). The local field of the propagating wave at the metal/dielectric boundary enhances absorption of plasmons as compared to free-space absorption. The subsequent fluorescent emission is out-coupled into free-space propagating lobes in accordance with the momentum matching conditions previously described. Fluorescence generated in this manner is emitted as directional lobes rather than omnidirectionally as in solution (i.e., as in a typical fluorometer). An optical detection system can be designed to capture directionally emitted fluorescence with much greater efficiency than can be done with omnidirectional fluorescence in a fluorometer. This enhanced capture efficiency results in considerably greater detection sensitivity and is sufficient to quantitatively measure cytokine secretion from single cells (see Reilly, M. T. et al. 2005, “SPR Surface Enhanced Fluorescence with a Gold-Coated Corrugated Sensor Chip” SPIE 6099-14TR, the entire disclosure of which is hereby incorporated by reference).